Rough Endoplasmic Reticulum- Structure and Function
Structure of the Rough Endoplasmic Reticulum (RER) ]The RER is an organelle that can be found almost all throughout the cell. It is composed of flat sealed sacs, called cisternae, that are continuous with the nuclear membrane (British Society for Cell Biology 2015). Electron microscopy shows these sheets of cisternae stacked on top of each other, making up the RER's structure (Shibata, Voeltz and Rapoport 2006). The stacked structure of the RER is depicted in Figure 3. What differentiates the RER from the Smooth Endoplasmic Reticulum (SER) is the fact it is studded with ribosomes. These ribosomes are membrane-bound and are on the RER's outer surface that interacts with the cytosol of the cell. This is the location of translocation of the proteins that enter the RER (British Society for Cell Biology 2015). The RER and SER are continuous domains, as shown in Figure 2. However, they do have segregated sheet and tubule structures- SER being tubular in most cases and RER being mostly flat and sheet-like (Shibata, Voeltz and Rapoport 2006). ] Functions of the Rough Endoplasmic Reticulum ''' '''Mechanism of Protein Synthesis and Translocation- An Important Function One of the most important functions of the rough endoplasmic reticulum (RER) is the synthesis of proteins on ribosomes and their translocation into the RER lumen. This is the mechanism of the process that allows the entry of these proteins: * A ribosome binds mRNA and synthesises protein in the cytoplasm. It synthesises proteins such as integral membrane proteins but not cytosolic proteins for example (Cooper and Hausman 2013). * During sequencing, a signal sequence is added to polypeptides amino terminus (Cooper and Hausman 2013). It is 11-27 amino acids long with a hydrophobic core surrounded by positively charged residues which forms an alpha helix (Fewell and Brodsky 2013). This signal sequence is recognised by a signal recognition particle (SRP) in the cytosol (Cooper and Hausman 2013). * The SRP is 6 polypeptides long and a 7S cytoplasmic RNA. **'FACT:' The RNA is highly conserved and thought to mediate SRP assembly and signal sequence recognition (Fewell and Brodsky 2013).** * The SRP binds the nascent polypeptide and ribosome, which halts further translation (Cooper and Hausman 2013). * The SRP binds the signal recognition particle receptor (SRPR) which associates the ribosome with a translocon (pore-like protein) on the ER membrane (Cooper and Hausman 2013). * When bound, the signal sequence is inserted into the translocon, and a conformational change occurs, opening this normally closed pore (Cooper and Hausman 2013). **FACT: In mammals, the translocon channels are complexes of 3 transmembrane proteins- Sec61 proteins (Cooper and Hausman 2013).** * The protein then enters the translocon and this causes the dissociation of the SRP the from the ribosome, allowing protein synthesis to resume and the protein feeds into the lumen of ER (Cooper and Hausman 2013). * Once in the lumen, the signal recognition sequence is removed by the signal peptide complex (SPC) and the protein binds to the chaperone which slows its folding (Braakman and Hebert 2013). **'FACT:' The slowed folding insures it is done correctly (Braakman and Hebert 2013).** This process is depicted in Figure 4 below. * But, for membrane-bound proteins with hydrophilic components, this does not occur. These proteins have hydrophobic/positively charged stop transfer regions (Cooper and Hausman 2013). The stop transfer region is a signal sequence that hasn’t been cleaved when it enters the ER (Fewell and Brodsky 2013). * This means the protein doesn’t pass through translocon as it recognises the stop transfer region, closing the channel and opens at the side, allowing lateral movement of the protein’s stop transfer region directly into the ER membrane (Cooper and Hausman 2013). * The proteins N terminus is luminal and the C terminus is cytosolic. If this orientation does not occur, the protein flips to ensure the termini are in the correct regions. This is directed by internal signal sequences that haven’t been cleaved on the protein (Cooper and Hausman 2013). This process is depicted in Figure 5 below. Folding proteins BiP is a member of the Hsp70 family (Braakman and Hebert 2013). It is the protein responsible for binding to the unfolded translated polypeptide as it enters the lumen of the ER through the Sec61 protein and folding it into the correct orientation before secretion to the Golgi apparatus (Cooper 2000). The BiP chaperone is made of two distinct domains: the nucleotide binding domain (NBD) and the substrate binding domain (SBD) (Braakman and Hebert 2013). When coupled to ATP, the NBD domain is in a low affinity state and doesn't bind to the substrate, however when the ATP is hydroylsed to ADP, the SBD domain binds to the nascent protein and prevents folding (Braakman and Hebert 2013). Nucleotide exchange factors (NEF) aid with the transition between the ADP and ATP bound states (Braakman and Hebert 2013). When ADP is phosphorylated back to ATP, the protein is folded into the correct orientation (Braakman and Hebert 2013). If the protein is not folded into the correct orientation, BiP remains bound and the polypeptide will be degraded (Cooper 2000). Protein disulfide isomerase PD1 breaks the incorrect disulfide bonds, largely the most accessible disulfide bonds within the unfolded polypeptide (Wilkinson and Gilbert 2004), and allows the formation of the correct sulfide bonds (Cooper 2000). This can only occur in the oxidising environment of the ER, as the reducing environment of the cytosol would support the -SH form of the bond (Cooper 2000). Reduction and oxidation will occur until the correct disulfide bond formation has been achieved (Wilkinson and Gilbert 2004). Peptidyl prolyl isomerase This enzyme is responsible for the isomerization of the cis form of the amino acid proline to the trans form, altering the function of the protein (Cooper 2000). The isomerization process is a very slow rate liming step, so the PPIase enzyme helps to speed up the isomerization (Braakman and Hebert 2013). Glycosylation On specific asparagine residues on the polypeptide, a common oligosaccharide (2-N-acetylglucosamine, 3 glucose, 9 mannose), synthesised on the lipid carrier, dolichol phosphate, is added (Cooper 2000). The asparagine must be in the sequence Asp-X-Ser or Asp-X-Thr (Cooper 2000). The N linked oligosaccharide is then modified further by removing 3 glucose residues and one mannose residue (Cooper 2000). The glycoprotein is then passed along to the golgi for further modification, before being translocated most likely to the plasma membrane to function as a signalling lipid (Cooper 2000). EXTRA LEARNING RESOURCES: If you would like to view a video that summarises this information and links the ER function to other organelles (the Golgi), here is a useful resource: https://www.youtube.com/watch?v=jDadorSbhi4